Diisocyanate conjugate and immunoassay characteristics influence detection of specific antibodies in HDI-exposed workers.

BACKGROUND: The structural characteristics of diisocyanate chemical protein antigens vary depending upon the methods of production, and may influence diisocyanate antigen immunoassays. The impact of different antigen preparation methods on immunoassay sensitivity, specificity, and predictive value for identifying workers with diisocyanate asthma (DA) has not been systematically evaluated. OBJECTIVE: Evaluate the influence of preparation methodology of hexamethylene diisocyanate human serum albumin (HDI-HSA) conjugates on the performance of specific antibody assays for identifying workers with confirmed HDI asthma. METHODS: Asthmatic reactions to HDI exposure were assessed in 80 autobody shop workers by specific inhalation challenge (SIC). HDI-specific IgE and IgG in serum were measured by RAST and ELISA with seven different HDI-HSA conjugates prepared in liquid phase with monomeric or polymeric HDI, or vapour-phase monomeric HDI. The HDI : HSA substitution ratios were determined by mass spectrometry. RESULTS: DA was confirmed by SIC in 23 subjects. The maximal sensitivity for detecting specific IgE among workers with positive SIC results was higher with RAST and with polymeric vs. monomeric HDI-albumin conjugates (21.7% vs. 8.7%) with a generally high specificity (>or=95%). HDI-HSA specific IgG antibody was also detected in 22-43% of HDI asthmatics depending upon the conjugate used. The specificity of specific IgG varied from 88% to 96%, and it was higher for monomeric (vs. polymeric) HDI-albumin conjugates with low (vs. high) substitution ratios. CONCLUSION: The test performance of specific IgE and IgG immunoassays for identifying a positive SIC response varied with different HDI-HSA conjugates. Standard test antigens and common immunoassays must be used to minimize inter-laboratory variability.

Exposure to protein aeroallergens in egg processing facilities.

Proteinaceous materials in the air can be highly allergenic and result in a range of immunologically mediated respiratory effects, including asthma. We report on the largest evaluation of exposure to date of airborne egg protein concentrations in an egg breaking and processing plant that had cases of occupational asthma. Personal air samples for egg protein were analyzed in duplicate on each PTFE filter using two analytical methods: (1) a commercial assay for non-specific total protein, and (2) indirect competitive inhibition assay using an ELISA method to quantify specific egg protein components. The highest concentrations were found in the egg washing room (mean exposure 644 microg/m3) and breaking room (255 microg/m3), which were also the areas where the risk of being sensitized was the greatest. There was excellent quantitative agreement between the airborne concentrations of total protein and sum of the specific protein antigens (ovalbumin, ovomucoid, and lysozyme). The correlation coefficient of the log-transformed data from the two methods was 0.88 (p < 0.0001). Size-selective sampling also indicated that most of the aerosol was capable of reaching the small airways. The methods described can be utilized to evaluate employee exposure to egg proteins. Exposure documentation, coupled with recommended exposure reduction strategies, could facilitate prevention of future employee sensitization and allergic respiratory responses by identifying high-exposure jobs and evaluating control measures.

Hypersensitivity pneumonitis in a metal-working environment.

BACKGROUND: An outbreak of lung disease among workers in a metal-working plant included 16 biopsy-confirmed cases of hypersensitivity pneumonitis and additional patients with asthma, bronchiolitis and emphysema, usual interstitial pneumonitis, and sarcoidosis. Study design Clinical examination of patients; cross-sectional questionnaire survey of the outbreak plant and two control plant areas, one with and one without MWF exposures, in a separate facility; industrial hygiene survey with laboratory characterization of microbial flora; and immunological investigation. METHODS: Patients with suspected hypersensitivity pneumonitis underwent a clinical examination including detailed lung function, imaging, and tissue studies. A plant walk-through identified metal-working processes, microbial aerosols, and work practices. Microbial characteristics of the three microbial aerosol-producing processes were characterized. Antibodies to those agents were determined in patient sera. A questionnaire survey was conducted in the case plant and in two areas of a control plant, one with and one without metal-working fluids exposure. RESULTS: Thirty-nine (79.6%) patients described symptoms consistent with work-related lung disease, eight received other diagnoses, and two did not complete their examinations. Sixteen patients had hypersensitivity pneumonitis confirmed on biopsy. Mean decrements in lung forced expiratory volume in 1 s and force vital capacity from before to after work were similar in the 16 biopsy-confirmed cases of hypersensitivity pneumonitis ( - 6.3%; - 7.2%) and the 19 symptomatic patients without biopsies ( - 11.2%, - 10.1%). Symptoms were more common in the case plant than in a non-MWF control plant area. Three sources of water-based aerosols were identified that grew similar microbial flora. Although machining increased airborne bacterial levels, the increase was not related to the concentration of viable bacteria in the sumps. Antibody testing did not identify a specific single organisms. Endotoxin levels were similar in case and MWF control plant. CONCLUSIONS: Lung disease in environments with water-based aerosols may be more common than usually recognized. Patients with HP often present with only subtle abnormalities and may be missed if multiple clinical abnormalities are required to document disease.

Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides.

Although health risks to pesticides containing Bacillus thuringiensis (Bt) have been minimal, the potential allergenicity of these organisms has not been evaluated. Therefore, a health survey was conducted in farm workers before and after exposure to Bt pesticides. Farm workers who picked vegetables that required Bt pesticide spraying were evaluated before the initial spraying operation (n = 48) and 1 and 4 months after (n = 32 and 20, respectively). Two groups of low- (n = 44) and medium- (n = 34) exposure workers not directly exposed to Bt spraying were also assessed. The investigation included questionnaires, nasal/mouth lavages, ventilatory function assessment, and skin tests to indigenous aeroallergens and to a variety of Bt spore and vegetative preparations. To authenticate exposure to the organism present in the commercial preparation, isolates from lavage specimens were tested for Bt genes by DNA-DNA hybridization. Humoral immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody responses to spore and vegetative Bt extracts were assayed. There was no evidence of occupationally related respiratory symptoms. Positive skin-prick tests to several spore extracts were seen chiefly in exposed workers. In particular, there was a significant (p < 0.05) increase in the number of positive skin tests to spore extracts 1 and 4 months after exposure to Bt spray. The number of positive skin test responses was also significantly higher in high (p < 0.05) than in low- or medium-exposure workers. The majority of nasal lavage cultures from exposed workers was positive for the commercial Bt organism, as demonstrated by specific molecular genetic probes. Specific IgE antibodies were present in more high-exposure workers (p < 0.05) than in the low and medium groups. Specific IgG antibodies occurred more in the high (p < 0.05) than in the low-exposure group. Specific IgG and IgE antibodies to vegetative organisms were present in all groups of workers. Exposure to Bt sprays may lead to allergic skin sensitization and induction of IgE and IgG antibodies, or both.

A cross-sectional survey of sensitization to Aspergillus oryzae-derived lactase in pharmaceutical workers.

BACKGROUND: The presence of IgE-mediated occupational respiratory sensitization to microbial enzymes has been well documented in a variety of industries. Aspergillus oryzae -derived lactase is used as a dietary aid for patients with lactose intolerance. OBJECTIVE: In 1993, a cross-sectional survey of 94 pharmaceutical workers exposed to lactase for a mean duration of 23 months and 24 nonexposed recently hired employees was initiated to identify lactase-sensitized workers and potential risk factors that could be used in making recommendations for preventing future cases of lactase sensitization. METHODS: The survey included a physician-administered questionnaire, skin prick testing to lactase enzyme and a panel of common aeroallergens, and spirometry. RESULTS: Twenty-seven of 94 lactase-exposed workers (29%) had positive skin test responses to lactase. These workers were 9 times more likely to have upper or lower respiratory symptoms compared with workers with negative skin test responses. Atopic workers were 4 times more likely to have lactase skin sensitivity than nonatopic workers. However, atopy was not a risk factor for the development of upper and/or lower respiratory symptoms. Lactase skin reactivity was not observed in the 24 nonexposed employees. CONCLUSION: This cross-sectional survey revealed that atopic workers were more likely to have lactase sensitization and that lactase-sensitized workers were more likely to have upper and/or lower respiratory symptoms, but atopy was not a risk factor for upper or lower respiratory symptoms. In spite of these findings, the company allowed only nonatopic, nonlactase-sensitized workers to continue working in high lactase-exposure areas with careful symptom monitoring and use of protective clothing. Although this strategy was successful in total prevention of new cases of occupational respiratory disease after 5 years, the results of this cross-sectional survey do not support exclusion of atopic workers from working with industrial enzymes.

Diisocyanate antigen-enhanced production of monocyte chemoattractant protein-1, IL-8, and tumor necrosis factor-alpha by peripheral mononuclear cells of workers with occupational asthma.

BACKGROUND: Previous studies have shown a significant association between confirmed diisocyanate-induced asthma (DOA) and in vitro production of diisocyanate antigen-stimulated histamine-releasing factors by PBMCs. Chemokines found in PBMC supernatants are known to express histamine-releasing factor activity. OBJECTIVE: PBMCs of diisocyanate-exposed workers were tested in vitro for diisocyanate antigen-specific enhancement of monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), macrophage inflammatory protein-1alpha, RANTES, IL-8, and T-cell cytokines that could play a regulatory role in chemokine synthesis (IL-4, IL-5, IFN-gamma, and TNF-alpha. METHODS: Secretion of chemokines and cytokines was determined by quantitative immunochemical assays of PBMC supernatants. Synthesis of mRNA for beta-chemokines was determined by reverse transcription-polymerase chain reaction. RESULTS: PBMCs of workers with DOA showed significantly enhanced secretion for MCP-1 compared with diisocyanate-exposed asymptomatic workers (P < .05). In vitro induction of antigen-stimulated MCP-1 mRNA synthesis in cultured PBMCs was demonstrated by reverse-transcription polymerase chain reaction. Quantitation of cytokines in supernatants showed increased mean production of IL-8 and TNF-alpha. IFN-gamma, IL-4, and IL-5 were not enhanced in subjects with DOA. CONCLUSION: Antigen stimulation of MCP-1 and TNF-alpha suggest that diisocyanate-specific cellular immune reactions result in activation of macrophages, which may be important in the pathogenesis of DOA.

T-cell receptor V beta gene segment expression in diisocyanate-induced occupational asthma.

BACKGROUND: Diisocyanates are the most common cause of occupational asthma induced by low-molecular-weight chemicals. The disease appears to be immunologically mediated but is independent of IgE antibody synthesis. An underlying genetic susceptibility is suggested by the fact that the disease only develops in approximately 5% to 10% of exposed workers. OBJECTIVE: The study was designed to determine whether disease susceptibility is influenced by HLA and T-cell receptor V beta gene segment usage. METHODS: T-cell receptor V beta repertoires were quantitated by using primer pairs specific for V beta gene segments in conjunction with a common C beta region primer. One group of workers with diisocyanate-induced occupational asthma produced diisocyanate-specific IgG and IgE antibodies, whereas the other group did not produce specific antibodies. Occupational asthma was previously confirmed by either workplace challenge or laboratory specific diisocyanate bronchoprovocation. Control groups consisted of diisocyanate-exposed workers who were free of symptoms, patients with nonoccupational asthma, and unexposed subjects who were free of symptoms. RESULTS: Lymphocytes from workers with diisocyanate-induced occupational asthma had significantly decreased V beta 1 and V beta 5 gene segment expression before in vitro exposure to diisocyanates, compared with control groups. Percent V beta 1 and V beta 5 gene segment expression was selectively increased when peripheral blood mononuclear cells were stimulated in vitro with diisocyanate-conjugated proteins. Low-resolution HLA class II phenotyping revealed no significant differences in the distribution of HLA-DR or HLA-DQ alleles between diisocyanate-induced occupational asthma and control groups. CONCLUSIONS: These findings are consistent with a hypothesis that antigen-specific T-cell subpopulations may be sequestered in the lungs of workers with diisocyanate-induced occupational asthma and clonally expand after further exposure to diisocyanates.

Characterization of histamine releasing factors in diisocyanate-induced occupational asthma.

Immunologic mechanisms contributing to diisocyanate-induced occupational asthma (OA) are poorly defined. There is a relatively low incidence of diisocyanate-specific IgE antibody responses. The frequent occurrence of delayed onset asthmatic responses in workers with diisocyanate asthma suggests a role for cellular immune mechanisms. We have shown in vitro production of antigen-specific mononuclear cell-derived histamine releasing factors (HRF) by peripheral blood mononuclear cells (PBMCs) of workers with OA. Monocyte chemoattractant protein-1 (MCP-1) and RANTES (acronym for "regulated on activation normal T expressed and secreted") are chemokines found in PBMC supernatants that express HRF activity. Diisocyanate-exposed workers were tested for diisocyanate antigen-stimulated enhancement of HRF, MCP-1, and RANTES production in supernatants of PBMCs and for serum specific IgE and IgG antibody levels to diisocyanate antigens bound to human serum albumin (HSA). PBMCs of workers with diisocyanate OA showed significantly increased production of antigen-specific HRF activity and MCP-1 ( > 300 ng/ml) compared to diisocyanate-exposed asymptomatic workers (P < 0.05). Antigen-stimulated enhancement of MCP-1 mRNA was demonstrated by reverse-transcription PCR. RANTES mRNA and chemokine secretion ( < 1 ng/ml) was also demonstrated in PBMCs, but did not show antigen enhancement in OA workers. Hapten specificity for the diisocyanate chemical to which a patient had been exposed was demonstrated for HRF enhancement and for IgG antibody reactions, but not for IgE reactions. HRF production was demonstrated in PBMC subpopulations, including lymphocytes and purified T cells. OA subjects showed increased CD8+ cells by immunofluorescence (mean CD4+: CD8+ = 1.2 +/- 0.2). The results suggest that diisocyanate antigen enhancement of HRF and MCP-1 production are stimulated by hapten-specific T cell reactions. Since a weak association has been found between IgE antibody synthesis and induction of diisocyanate OA, the role of T cell cytokines and chemokines in the pathogenesis of OA requires further investigation.

Occupational asthma caused by FD&C blue dye no. 2.

We report the case of a 55-year-old male who experienced cough, dyspnea, wheezing, and nasal congestion immediately upon exposure to FD&C Blue Dye No. 2 (Indigotine) at work. The patient had worked for 10 years mixing and grinding powdered synthetic red, yellow, and blue dyes for use in foods; symptoms had occurred for 2 years and only with exposure to Indigotine (C16H8N2Na2O8S2), a free flowing blue powder. Prick testing to Indigotine (20 mg/mL) was negative. ELISA failed to detect specific IgE, IgA, IgM, or IgG to Indigotine-HSA conjugates. Bronchial challenge was done according to the method of Pepys et al. beginning with 4 x 10(-4) lactose dilution of Indigotine powder. After 5 minutes of exposure to 4 gm Indigotine/100 gm lactose, the patient developed dyspnea and audible wheezing. At 20 minutes postexposure, there was a 20% decline in FEV1 from prechallenge baseline; no late phase response was observed. A second bronchial challenge with sodium sulfate, the major nondye product additive was negative. To our knowledge, this is the first documented case of occupational asthma due to FD&C Blue Dye No. 2. The pathogenesis is uncertain but does not appear to be IgE mediated.

Machine operator's lung. A hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols.

Six auto parts manufacturing workers were referred for evaluation of a 6-week history of work-related dyspnea, cough, and fatigue. Two workers also reported fever and weight loss. All six worked in a machining area where a waterbased metalworking fluid was used and recirculated under high pressure, thereby creating an aerosol. Chest radiographs revealed pulmonary interstitial infiltrates in four workers. Lung function tests showed that four workers had decreased diffusing capacity. After removal from the work area, all workers recovered. The metalworking fluid was cultured for bacteria and fungi. Isolates from broth cultures were sonicated to obtain antigen extracts. Serum precipitins to one or more of the microbial isolates were identified in all six workers but not in eight of nine nonexposed control subjects. The most frequent precipitin response (six of six workers) was against antigens of Pseudomonas fluorescens, which was cultured from the metalworking fluid. In all workers, precipitins to at least one other cultured organism were detected; these included Aspergillus niger, Staphylococcus capitas, an acid-fast Rhodococcus sp, and Bacillus pumilus. This represents the first report of hypersensitivity pneumonitis associated with industrial exposure to aerosolized metalworking fluid. Observed precipitin responses to a variety of microbial contaminants in metalworking fluid strongly suggest a causative role for microbial antigens in the induction and elicitation of this manifestation of hypersensitivity pneumonitis.

Modulation of T-cell ontogeny by transplacental benzo(a)pyrene.

Transplacental exposure to the carcinogen, benzo(a)pyrene BaP, leads to depressed immune function and increased tumor incidence in mice. This paper reports ontogenetic T-cell changes in BALB/c mice after exposure to BaP in utero. Monoclonal antibodies (MAbs) were produced to fetal liver T-cells (FLT) and newborn spleen (NBS) lymphocytes purified from offspring of pregnant BALB/c mice that were given one injection of BaP (150 mg/kg body weight) in mid-gestation (day 11-13). The MAbs reacted with two T-cell membrane antigens (FLT and NBS) found in fetal liver, neonatal and adult thymus and spleen. Lymphocytes of BaP-exposed 19-day fetuses showed decreased subpopulation frequencies (P < 0.05) in fetal liver total T-cells (from 56% to 16%), Ly1 cells (from 33% to 9%), and Ly2 cells (from 56% to 1%) compared with untreated controls. In contrast, BaP increased the subpopulation frequencies (P < 0.05) in FLT cells in fetal liver (from 20% to 52%) and in newborn spleen (from 21% to 51%), and increased NBS cells in newborn spleen (from 24% to 59%). The increased frequency in FLT and NBS cells was due to their relative resistance to BaP toxicity and/or BaP-enhanced proliferation in the neonatal period. Compared with untreated controls, BaP treatment resulted in reduced numbers of T-cells in fetal liver and showed a selective toxicity for Ly1 cells (89% reduction) and Ly2 cells (99% reduction), whereas FLT cells were not reduced and NBS cells were reduced by 60%. Six-week-old juvenile mice exposed to BaP in utero showed recovery of total T-cells to control levels in spleen and thymus, but showed depletion (P < 0.01) in thymic FLT cells (from 81% to 12%) and in splenic NBS cells (from 55% to 16%). The monoclonal antibodies developed for this study recognize novel cellular changes in the murine immune system that are associated with transplacental BaP. The FLT and NBS antigens may be useful biomarkers for developmental immune dysfunctions in progeny exposed to BaP in utero.

Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles.

This paper describes the trapping of antigen in lymphoid follicles of rat popliteal lymph nodes as revealed by electron microscopic radioautographs following injection of (125)I-labeled Salmonella adelaide flagella and other materials. The antigen was taken up vigorously, and to an approximately equal extent, by both primary and secondary follicles. The rate of uptake was faster in preimmunized than in virgin adult rats. The bulk of the antigen in follicles was extracellular, and persisted in this location for at least 3 wk. Label was most frequently found at or near the surface of fine cell processes. Many of these were branches of dendritic follicular reticular cells. Such processes interdigitated with equally fine processes of lymphocytes, creating an elaborate meshwork. In some cases, antigen was found between lymphocytes which appeared to be in close apposition. Occasionally, a few grains appeared over lymphocyte nuclei and study of serial sections suggested that this probably represented true entry of small amounts of antigen into lymphocytes. The characteristic "tingible body" macrophages (TBM) of germinal centers appeared to play only a secondary role in follicular antigen retention. They showed degrees of labeling over their phagocytic inclusions varying from negligible to moderately heavy. Moreover, follicles lacking or poor in TBM retained antigen just as effectively as those containing numerous TBM. The hypothesis is advanced that TBM may be derived from monocytes that migrate down from the circular sinus. Follicular localization of three other materials was also studied, though not in such detail. These were (125)I-HSA complexed to anti-HSA: (125)I-labeled autologous IgG; and (125)I-monomeric flagellin. All of these showed the basic features of intercellular, membrane-associated deposition noted with (125)I-flagella. The role of follicular antigen depots in immune induction is discussed. The tentative conclusion is reached that follicular antigen in a primary follicle encounters natural antibody on the surface of certain antigen-reactive lymphocytes. The resultant reaction causes blast cell transformation and eventually the genesis of a germinal center.